Borrelia burgdorferi (Bb), the Lyme disease spirochete, undergoes dramatic adaptive changes as it cycles in nature between its diverse tick and mammalian hosts. In 2001, we discovered that the alternative sigma factor RpoS (? controls the synthesis of key outer membrane lipoproteins that confer mammalian infectivity and pathogenicity to Bb. We additionally discovered that RpoS itself is controlled by another alternative sigma factor, RpoN (? ?), that binds to the rpoS promoter. We named this novel pathway in Bb the RpoN-RpoS regulatory pathway. This pathway is the first example of an alternative sigma factor regulatory cascade controlling virulence in a bacterial pathogen. The RpoN-RpoS pathway is activated in Bb by various environmental stimuli that accompany tick feeding, and is sustained during Bb's mammalian infection phase. Over the past funding interval, we made the additional striking discovery that in addition to the enhancer- binding protein (Rrp2) needed for RpoN activation, another protein known as BosR (BB0647) functions as a second activator to promote RpoN-dependent rpoS transcription in Bb. BosR is the first example, in any bacterium, of an additional activator essential for ?-dependent gene transcription. This system represents a new paradigm of ?-mediated gene control in bacteria. We have further determined that BosR is a Zn+2- containing DNA-binding protein that binds to a core sequence (BosR box) of ATTTAANTTAAAT. Bioinformatics and in vitro electrophoretic mobility shift assays have revealed three potential BosR binding sites within rpoS, one of which is located immediately adjacent to the RpoN binding site. However, there remain many unanswered questions concerning how BosR functions, particularly when Bb is in its native environments of ticks and mammals. In Aim 1 of this proposal, we shall assess the biological role(s) of the three BosR binding sites in rpoS. Selected rpoS promoter mutations/deletions will be examined for their influence on rpoS expression in growing Bb, and selected rpoS promoter mutants also will be assessed for their ability to escape from ticks and infect and disseminate in mice. In Aim 2, we shall structurally character BosR, using both NMR spectroscopy and X-ray crystallography, to examine parameters explaining how BosR coordinates metal, has a predilection to form homodimers, participates in protein-protein interactions, and how it binds to DNA conformationally. In Aim 3, we shall determine the regulons influenced by BosR, RpoN, and RpoS when Bb is within its native settings of ticks and mammals; these have not been previously compared directly. New gene targets of interest also will be assessed for their roles in the life cycle of Bb. These combined studies will (i) provide new insights into potentially blocking the transmission of Bb, (ii) clarify the novel mechanism by which BosR activates the central RpoN-RpoS pathway or other RpoN-independent genes for Bb's virulence; and (iii) further elucidate this new paradigm of ?-mediated bacterial gene control. Resultant findings could lead to new intervention strategies (vaccines, therapeutics) for Lyme disease.